Casein Gene Promoter Research Articles

Functional analysis of the bovine beta- and kappa casein gene promoters using homologous mammary gland derived cell line

Abstract. Bovine casein gene cluster belongs to the best studied regions of the bovine genome. However, molecular basis of the regulation of casein gene expression is still of great interest for the advancement of milk production. Identification of crucial regulatory regions governing casein gene expression would provide valuable information for marker assisted selection in dairy cattle. In our study we performed comparative analysis of the bovine beta- and kappa casein gene promoter sequences with the regulatory sequences from some other species. In addition, we used homologous mammary gland derived cell culture and luciferase reporter gene system to confirm the functionality of the proximal beta and kappa casein promoters. The longer kappa casein promoter (2064 bp) showed the highest expression level, followed by the short kappa casein promoter (925 bp) and beta casein promoter (1692 bp). Here we demonstrate the suitability of the bovine mammary gland derived cell line BME UV1 for transient gene expression under transcriptional control of the bovine casein gene promoters and compare functionality of different fragments of bovine beta- and kappa casein gene promoters using homologous in vitro system.

Functional study of the equine β-casein and κ-casein gene promoters

Casein genes are expressed in a tissue-specific and highly coordinated manner. The main goals of casein gene promoter studies are to unravel cis- and trans-acting factors involved in the complex signalling pathway controlling milk production, and to explore the possibility of using these promoters for tissue-specific production of heterologous proteins in the mammary gland. Here we present a comparative study of the equine β-casein and κ-casein gene proximal promoters. In order to confirm the assumption that in the horse, as in other mammalian species, casein genes are organized in a cluster located on a single chromosome, we performed in situ hybridization of pro-metaphase chromosomes with two BAC clones containing different equine casein genes. Sequence analysis of the β-casein and κ-casein gene proximal promoters revealed binding sites for activators (STAT5, GRE, NF1, MAF) and repressors (YY1, PMF), characteristic for casein genes. The alignments of casein gene promoters revealed the highest sequence identity in the proximal promoter region between the equine and human β-casein gene promoters. We directly compared the activity of equine β-casein and κ-casein gene promoters in vitro using bovine mammary gland cell line BME-UV1. In this system, the κ-casein gene proximal promoter activated the reporter gene expression more efficiently than the β-casein gene promoter of approximately the same length. The 810 bp of β-casein promoter activated the reporter gene expression more efficiently than the long fragment (1920 bp) and the 1206 bp fragment of the same promoter, which included also 396 bp of 5′ UTR.

Association of nucleotide-sequence polymorphism in the 5’‑flanking regions of bovine casein genes with casein content in cow’s milk

The 5'-flanking regions (promoters) of the bovine αs1-, αs2- and β-casein genes were analysed for DNA sequence variants using PCR/RFLP in Polish Red (PR) and Black-and-White (BW) cattle. The polymorphic sites occurred at positions -1084 and -186 in the promoter region of the αs2-casein gene and at -728 and -109 in the αs1- and β-casein genes, respectively. These poly- morphic sites were located within known potential regulatory sequences, suggesting an influence on the binding of transcription factors and expression of the bovine casein genes. Associations were found between various genotypes in the promoter region of the αs2-casein gene and αs1- and β-casein genotypes, thus showing the existence of intergenic haplotypes within the bovine casein locus. Milk proteins derived from cows of varying genotypes in the casein gene promoters were analysed using SDS-PAGE and HPLC techniques. It was shown that nucleotide sequence polymorphism in the pro- moter region of the bovine αs2-casein gene was associated with various contents of αs2- and β-casein in the milk. Bovine casein gene / 5'-flanking region / gene polymorphism / milk protein

The first intron of the murine β-casein gene contains a functional promoter

Caseins are the major milk proteins in most mammals. Together with calcium and phosphate they form the casein micelle. The corresponding casein genes are clustered in mammalian genomes and their expression is coordinately regulated with regard to developmental and tissue specificity. Casein gene promoters are responsive to lactogenic hormones, cell–matrix, and cell–cell interactions. Transcriptional enhancer elements are found in the 5 ′ upstream regions of casein genes but have also been detected in the first intron of the bovine β-casein gene. We show here that the first intron of the murine β-casein gene has three discernible functions. First, transcriptional enhancer elements present in the intron increase the basal activity of the β-casein promoter. In addition, these intronic enhancer elements augment the induction of the β-casein promoter by lactogenic hormones. Finally, we demonstrate that the first intron of the murine β-casein gene contains a functional promoter.

Involvement of Oct-1 in transcriptional regulation of β-casein gene expression in mouse mammary gland

Mouse β-casein gene promoter contains a region termed block C which is crucial for its gene transcription induced by lactogenic hormones. Nuclear extracts from mouse mammary glands contain at least two binding complexes (DS1 and DS2) which specifically bind to double-stranded block C region DNA. The binding sequence of these complexes was identified to be 5′-AAATTAGCATGT-3′ which contains a sequence element related to the consensus octamer motif's complement ATTTGCAT. In the present study, we demonstrate that this sequence element indeed is the binding site for octamer-binding transcription factors (Octs) and Octs represent the double-stranded DNA binding proteins specifically binding to the block C region. Formation of the specific double-stranded binding complexes can be completely blocked by Oct binding motif oligonucleotides and anti-rOct-1 antiserum. We also show that Oct-1B represents at least partial, if not all, double-stranded binding protein, DS1, in mammary nuclear extract. Oct-1B may function as a transcriptional activator on casein gene promoter. The Oct binding activity to beta-casein gene promoter in the mammary gland is affected under influence of hormones both in vitro and in vivo. The DS1 binding activity can be induced by the combination of lactogenic hormones insulin, hydrocortisone and prolactin in organ culture of virgin mouse mammary gland. The binding activity in vivo can be induced by injection of progesterone or its combination with estradiol in virgin mice.

The milk protein promoter is a useful tool for developing a rat with tolerance to a human protein.

Biopharmaceuticals intended for humans are immunogenic in animals. Antibodies associated with their administration make it difficult to perform repeated-dose pharmacology and toxicology studies in animals. Despite suggestions to solve this problem with transgenic animal technology, an effective strategy has not yet been reported. The objective of the present study was to provide an efficient strategy to develop rats tolerant to biopharmaceuticals such as human gene-based proteins. The present study used transgenic rat lines (lines 311-6, 308-5, and 305-1) carrying a fusion gene designed to express the human growth hormone (hGH) gene under the control of the bovine alphaS1 casein gene promoter. Three lactating females with the transgene, produced approximately 4 mg/ml, 300 microg/ml, and 10 ng/ml in their milk. Male 8-week-old rats from these three lines were immunized with hGH three times (week 0, 1, and 3 ) and the production of antibodies against hGH in their sera were examined at week 4. While the hGH serum antibody titers increased over 1000-fold in wild-type control rats, there was no detectable antibody against hGH in the sera of these three transgenic lines. Human growth hormone in their sera was undetectable (lines 308-5 and 305-1) or much lower than the endogenous biologic level of rat growth hormone (line 311-6). Importantly, lines 308-5 and 305-1 developed tolerance to hGH without detectable hGH in their sera and these lines will be very useful for the repeated dose pharmacology and toxicology studies. These results suggest that a milk protein promoter can be a useful tool to develop transgenic rats that are tolerant to biopharmaceuticals intended for humans.

Transgene expression in mammary glands of newborn rats.

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals.

Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression.

A highly conserved CCAAT/enhancer-binding protein (C/EBP)-binding site centered around -134 relative to the transcription start site in the rat beta-casein gene promoter is capable of interacting specifically with recombinant and mammary gland C/EBP proteins. Western blot analysis indicates that C/EBP levels change dramatically throughout mammary gland development. C/EBP alpha expression is barely detectable in mammary glands from virgin and pregnant animals but is expressed at high levels during lactation and at lower levels during involution. The expression of three C/EBP beta isoforms [the liver-enriched activating proteins (LAPs); and the liver-enriched inhibiting protein (LIP)] is elevated throughout pregnancy, with LIP expression increasing more than 100-fold. Thus, during pregnancy, a low LAP/LIP ratio (< 5) is maintained. C/EBP beta expression decreases at parturition, with LIP diminishing to levels observed in the virgin gland. Therefore, during lactation a more than 100-fold increase in the LAP/LIP ratio is observed. Treatment of the HC11 mammary epithelial cell line with hydrocortisone results in a 10- to 20-fold inhibition of LIP expression, with only minor changes in LAP levels. Therefore, glucocorticoids may impinge upon beta-casein gene expression by altering the ratio of the inhibitory to the activating isoforms of C/EBP beta. Several previously defined casein gene promoter regions capable of conferring hormone and extracellular matrix inducibility to reporter genes in mammary cells are suggested to be composite response elements, containing putative binding sites for the same set of hormonally and developmentally regulated factors: C/EBP, MGF/Stat5, and the glucocorticoid receptor.

Cortisol induces rapid accumulation of whey acid protein mRNA but not of as1 and b-casein mRNA in rabbit mammary explants

Mammary explants from rabbit were cultured in the presence of various combinations of insulin, cortisol and prolactin. The concentration of whey acidic protein (WAP) as1-casein and b-casein mRNA was measured using specific cDNA probes. Medium alone was added to the explants for one day. Prolactin with and without cortisol was then added to the medium. Prolactin alone induced rapidly as1-and b-casein gene but not WAP gene. When cortisol was added with prolactin, the as1-and b-casein genes were induced at the same rate as in the absence of the steroid. In contrast, the WAP mRNA was then rapidly accumulated. This induction process was not altered by cycloheximide for two hours and it was blocked at a later stage. In a second experiment, insulin and prolactin were first added for 24 hours in the culture medium. Cortisol was then added and the concentration of the three mRNA was measured. Cortisol did not significantly modify the level of as1-and b-casein mRNA. On the contrary, the WAP mRNA was rapidly accumulated. These data indicate that the well-established amplificatory effect of glucocorticoids on casein gene expression is a slow process whereas their effect on the WAP gene is rapid. This suggests that glucocorticoids induce casein gene expression through an indirect cellular mechanism not involving a glucocorticoid receptor element in casein gene promoters and that WAP gene is more classically stimulated through the direct binding of the steroid receptor to a glucocorticoid receptor element located in its promoter.